Fungal stress proteins

ABSTRACT

A polypeptide sequence from Candida albicans is described which has significant sequence homology with known stress proteins from other organisms, particularly the heat shock protein hsp 90 of Saccharomyces cerevisiae. Corresponding DNA sequences are also described, together with antibodies raised against fragments of the sequence. The polypeptide and DNA sequences and antibodies provide separate means for the diagnosis and/or treatment of fungal, particularly Candida, infections.

This is a division of application Ser. No. 8/357,264 filed Dec. 13,1994, now U.S. Pat. No. 5,541,077 which is a continuation of Ser. No.08/152,669, Nov. 16, 1993 now abandoned; which was a divisional of Ser.No. 07/663,897 filed as PCT/GB90/1021 Jul. 2, 1990, now U.S. Pat. No.5,288,639 issued Feb. 22, 1994.

FIELD OF THE INVENTION

This invention relates to fungal stress proteins, to corresponding DNAsequences, to fungal stress protein inhibitors and to their use inmedicine and for diagnosis.

BACKGROUND OF THE INVENTION

Environmental stress can induce an increase in the rate of synthesis ofso-called heat shock, or stress, proteins in both procaryotic andeucaryotic cells see for example Schlesinger et al (eds) in Heat Shockfrom Bacteria to Man, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1972)!. Although the function of stress proteins has yet to befinally resolved, some have been reported to participate in assembly andstructural stabilization of certain cellular and viral proteins, andtheir presence at high concentration may have an additional stabilisingeffect during exposure to adverse conditions.

Many pathogenic organisms have been shown to produce stress proteins seefor example Young D, et al., Proc. Natl. Acad. Sci. USA 85, 4267-4270(1988)!. The proteins are thought to be produced in response to thestress of infection to help protect the invading pathogen. Thus, forexample, the ability to produce stress proteins has been implicated inthe survival of bacterial pathogens within macrophages Christmas, M. F.et al. Cell, 41, 753-762 (1985) and Morgan, R. W. et al, Proc. Natl.Acad. Sci. USA, 83, 8059-8063, (1986)!.

It has been suggested that the presence of stress proteins in a varietyof human pathogens indicates that the stress response is a generalcomponent of infections, and that stress proteins should be consideredamong candidates for subunit vaccines Young, D. et al, ibid!. Candidaalbicans is, medically, the most important of the human fungalpathogens. Systemic candidiasis (candidosis) is an increasingly commoncause of death amongst immunocompromised and debilitated patients, witha mortality of over 70% Gold, J.W.M., Am. J. Med. 76, 458-463, (1984)!;while oral candidiasis is a frequent early manifestation of the acquiredimmunodeficiency syndrome Klein, R. S. et al, N. Engl. J. Med. 311,354-357, (1984)!. Candidiasis is difficult to diagnose, and is not easyto treat, mainly since the usual method of treatment involves use ofamphotericin B, which is itself highly toxic. A need therefore exists inthe diagnosis and treatment of Candida infections for more sensitivediagnostic methods and treatment which has less toxic side effects.

A number of Candida antigens have been detected in the sera of patientswith systemic candidiasis Matthews, R. C. et al, J. Clin. Microbiol. 25,230-237 (1987)!. One of these, with a relative molecular mass ofapproximately 47 kilodaltons (47 kd) is an immunodominant antigen whichhas been reported by four independent groups Matthews R. C., et alLancet ii, 1415-1418 (1984); Au-Young, JK et al, Diagn. Microbiol.Infect. Dis., 3, 419-432, (1985); Neale T. J., et al, Aust. N. Z. J.Med., 17, 201-209 (1987); and Ferreira R. P. et al, J. Clin. Microbiol.28, 1075-1078 (1990)!. This 47 Kd antigen is distinct from the 48-52 Kdantigen described by Strockbine et al Strockbine N. A. et al Infect.Immun. 43, 715-721 (1984)! for two reactions:

(1) monoclonal antibodies raised against the 48-52 Kd antigencross-react with antigens at 120-135 Kd and 35-38 Kd Strockbine N. A. etal, Infect Immun 43, 1012-18 (1984); Buckley H. R., et al U.S. Pat. No.4,670,382 (1987)! and

(2) the 48-52 Kd antigen it is an enolase Safranek W. W. and Buckley H.R., Second Conference on Candida and Candidiasis, Abstract A7 (1990)!.In contrast, antibody to the 47 Kd antigen cross-reacts with an antigenat about 92 Kd (see below). An immunodominant C. albicans antigen of54.3 Kd (range 48.9 to 59.7 Kd) was described by Greenfield R. A., andJones J. M., in Infect. Immun. 34, 469-477 (1981).

SUMMARY OF THE INVENTION

We have now been able to clone and express part of the DNA sequenceencoding the 47 Kd antigen, and in doing so we have surprisinglydiscovered a polypeptide sequence which has significant sequencehomology with known stress proteins from other organisms, particularlythe heat shock protein hsp 90 of Saccharomyces cerevisiae Farrelly F. W.and Finkelstein D. B., J. Biol. Chem. 259, 5745-5751 (1984)!. InSaccharomyces two genes exist, hsp 90 and hsc 90, coding for 98%homologous proteins Finkelstein D. B. and Farrelly F. W., Fed. Proc. 43,1499 Abstr. 482 (1984)!. Either of the genes can be deleted withoutaffecting cell viability, however, deletion of both genes is lethal. Theseparate genes produce a constitutive and an inducible form D.Finkelstein, personal communication cited in Lindquist S. Ann. Rev.Biochem. 55, 1151-91 (1986)!.

We can also now suggest that there are two forms of a C. albicans stressor heat shock protein hsp90. Thus a 47 Kd breakdown product occurs onimmunoblots of C. albicans grown at 37° C. or 23° C. whereas a 92 Kdantigen only appears at 37° C. This suggests a heat-inducible, stablehsp 90 is produced at 37° C., but not at 23° C., whereas a more labilehsp 90 occurs at 23° C. which breaks down to the 47 Kd subcomponentunder denaturing conditions. We have used this discovery to developmeans for the improved diagnosis and treatment of candida infections andrelated fungal disease.

Thus according to one aspect of the invention we provide a fungal stressprotein having an amino acid sequence which includes at least thesequence of formula (1) (SEQ ID NO:1). ##STR1## or a fragment thereof,or an analogue thereof.

The single letters in formula (1) are each to be understood to representa separate amino acid, and each is the conventional single letter symbolused for amino acids.

The stress protein according to the invention may be of fungal originand may be obtainable, for example, from strains belonging to thegeneral Candida, for example Candida parapsilosis, Candida krusei and,in particular, Candida albicans and Candida tropicalis; Cryptococcus,for example Cryptococcus neoformans; Histoplasma, for exampleHistoplasma capsulatum, and related yeasts; and Aspergillus, for exampleAspergillus fumigatus and related filamentous fungi.

Particular fragments of a stress protein according to the inventioninclude any peptide epitopes, for example of a few amino acids oranalogues thereof. Examples of such epitopes include STDEPAGESA (SEQ IDNO:4), LSREM (SEQ ID NO:5), LKVIRK (SEQ ID NO:6) and LKVIRKNIVKKMIE (SEQID NO:7). Peptides of this type may be synthesized using conventionalliquid or solid phase peptide synthesis techniques.

Analogues of a stress protein according to the invention include thoseproteins wherein one or more amino acids in the sequence of formula (1)is replaced by another amino acid, providing that the overallfunctionality of the protein is conserved.

A stress protein according to the invention may be obtained in apurified form, and thus according to a further aspect of the inventionwe provide a substantially pure fungal stress protein having an aminoacid sequence which includes at least the sequence of formula (1), andanalogues thereof.

The term substantially pure is intended to mean that the stress proteinaccording to the invention is free from other proteins of fungal origin.In the various aspects of the invention described hereinafter it is tobe understand that a reference to the fungal stress protein alsoincludes substantially pure preparations of the protein.

In a further aspect the invention particularly provides a recombinantfungal stress protein having an amino acid sequence which includes atleast the sequence of formula (1) or a fragment thereof, or an analoguethereof.

A stress protein according to the invention may be further characterisedby one or more of the following features:

(1) it has an isoelectric point (pI) in a range around pI 4 to pI 5;

(2) it is an immunodominant conserved antigen;

(3) patients recovering from systemic candidiasis seroconvert to thestress protein;

(4) patients with acquired immunodeficiency syndrome have antibody tothe stress protein;

(5) it cross-reacts with the 47 kilodalton and approximately 92kilodalton antigens of Candida albicans using affinity-purifiedpolyclonal monospecific antibody against the 47 kilodalton antigen.

A stress protein according to the invention has a number of uses. Thus,for example, the protein may form the basis of a diagnostic test forfungal infection, for example an immunological test such as anenzyme-linked immunosorbent assay, a radioimmunoassay or a latexaggulutination assay, essentially to determine whether antibodiesspecific for the protein are present in an organism.

In another use, the stress protein according to the invention may beemployed, using conventional techniques, for screening to obtainactivity inhibiting agents for use in the treatment of fungalinfections. Such a screening method forms a further aspect of theinvention.

In a further use, the stress protein according to the invention isparticularly well suited for the generation of antibodies. Thusaccording to a further aspect of the invention we provide a fungalstress protein having an amino acid sequence which includes at least thesequence of formula (1) or a fragment thereof or an analogue thereof,for use as an immunogen.

Standard immunological techniques may be employed with the stressprotein in order to use it as an immunogen. Thus, for, example, anysuitable host may be injected with the protein and the serum collectedto yield the desired polyclonal anti-stress protein antibody afterpurification and/or concentration. Prior to injection of the host thestress protein may be formulated in a suitable vehicle ad thus accordingto a further aspect of the invention we provide a composition comprisinga fungal stress protein having an amino acid sequence which includes atleast the sequence of formula (1) or an analogue thereof together withone or more pharmaceutically acceptable excipients.

For purification of any anti-stress protein antibody, use may be made ofaffinity chromatography employing an immobilised stress protein of theinvention as the affinity medium. This according to another aspect ofthe invention we provide a fungal stress protein having an amino acidsequence which includes at least the sequence of formula (1), or a partthereof or an analogue thereof, covalently bound to an insolublesupport.

The use of the stress proteins according to the invention as immunogensfor the production of antibodies generates one type of inhibitor of theaction of the protein. Generally, inhibitors of the stress proteins arepotentially useful in the diagnosis, and in particular the treatment, offungal infections and provide a further feature of the invention.Inhibitors include any antagonists of the action of the stress proteinsor agents which prevent their production, and in particular includethose which may be used in the treatment of fungal infections. Suitableinhibitors include for example pharmaceutical reagents, includingantibodies, and chemical analogues of the stress proteins to antagonisethe action of the stress protein, and anti-sense RNA and DNA to preventproduction of the stress protein. Suitable inhibitors may be determinedusing appropriate screens, for example by measuring the ability of apotential inhibitor to antagonise the action of, or prevent theproduction of a stress protein according to the invention or a fragmentthereof, or an analogue thereof, in a test model for example an animalmodel such as the mouse model described in the Examples hereinafter.

According to a further aspect of the invention we provide an inhibitorof a fungal stress protein, said protein having an amino acid sequencewhich include at least the sequence of formula (1) or a fragment thereofor an analogue thereof, for use in the diagnosis or treatment of fungalinfections.

Inhibitors may be used either along or where appropriate in combinationwith other pharmaceutical agents, for example, other anti-fungal agents,such as amphotericin or flucytosine.

One particularly useful group of inhibitors according to this aspect ofthe invention are antibodies capable of recognising and binding to thestress proteins.

Thus, according to yet another aspect of the invention we provide anantibody specific for one or more epitopes of a fungal stress proteinhaving an amino acid sequence which includes at least the sequence orformula (1) or a fragment thereof or an analogue thereof.

The antibody may be a whole antibody or an antigen binding fragmentthereof and may in general belong to any immunoglobulin class. Thus, forexample, it may be an immunoglobulin M antibody or, in particular, animmunoglobulin G antibody. The antibody or fragment may be of animal,for example mammalian origin and may be for example or murine, rat orhuman origin. It may be a natural antibody or a fragment thereof, or, ifdesired, a recombinant antibody or antibody fragment, i.e. an antibodyor antibody fragment which has been produced using recombinant DNAtechniques.

Particular recombinant antibodies or antibody fragments include, (1)those having an antigen binding site at least part of which is derivedfrom a different antibody, for example those in which the hypervariableor complementarity determining regions of one antibody have been graftedinto the variable framework regions of a second, different antibody (asdescribed in European Patent Specification No. 239400); (2) recombinantantibodies or fragments wherein non-Fv sequences have been substitutedby non-Fv sequences from other, different antibodies (as described inEuropean Patent Specifications Nos. 171496, 173494 and 194276); or (3)recombinant antibodies or fragments possessing substantially thestructure of a natural immunoglobulin but wherein the hinge region has adifferent number of cystein residues from that found in the naturalimmunoglobulin, or wherein one or more cysteine residues in a surfacepocket of the recombinant antibody or fragment is in the place ofanother amino acid residue present in the natural immunoglobulin (asdescribed in International Patent Applications Nos. PCT/GB 88/00730 andPCT/GB 88/00729 respectively).

The antibody or antibody fragment may be of polyclonal, or preferably,monoclonal origin. It may be specific for a number of epitopesassociated with the stress protein but it is preferably specific forone.

Antigen binding antibody fragments include for example fragments derivedby proteolytic cleavage of a whole antibody, such as F(ab')₂,Fab' or Fabfragments, or fragments obtained by recombinant DNA techniques, forexample Fv fragments (as described in International Patent ApplicationNo. PCT/GB 88/00747).

The antibodies according to the invention may be prepared usingwell-known immunological techiques employing the stress protein asantigen. Thus, for example, any suitable host may be injected with thestress protein and the serum collected to yield the desired polyclonalantibody after appropriate purification and/or concentration (forexample by affinity chromatography using the immobilised stress proteinas the affinity medium). Alternatively splenocytes or lymphocytes may berecovered from the stress protein injected host and immortalised usingfor example the method of Kohler et al, Eur. J. Immunol. 6, 511, (1976),the resulting cells being segregated to obtain a single genetic lineproducing monoclonal anti-fungal stress proteins. Antibody fragments maybe produced using conventional techniques, for example by enzymaticdigestion, e.g. with pepsin (Parham, J. Immunol, 131, 2895 (1983)! orpapain Lamoyi and Nigonoff, J. Immunol. Meth., 56, 235, (1983)!. Whereit is desired to produce recombinant antibodies according to theinvention these may be produced using for example the methods describedin European Patent Specifications Nos. 171496, 173494, 194276 and239400.

Antibodies according to the invention may be labelled with a detectablelabel or may be conjugated with effector molecule for example a druge.g. an anti-fungal agent such as amphotericin B or flucytosine or atoxin, such as ricin, or an enzyme, using conventional procedures andthe invention extends to such labelled antibodies or antibodyconjugates.

The antibodies according to the invention have a diagnostic and/ortherapeutic use. Thus for diagnostic use the antibodies may be employedto detect whether the stress protein is present in a host organism, toconfirm whether the host has a particular fungal infection, for examplean infection due to a Candida, Cryptococcus, Histoplasma or Aspergillusorganism, for example in the diagnosis of fungal abcesses, especiallyhepatic Candidiasis, and/or to monitor the progress of therapeutictreatment of such infections. Diagnostic methods of this type form afurther aspect of the invention and may generally employ standardtechniques, for example immunological methods such as enzyme-linkedimmunosorbent methods, radioimmuno-methods, lates agglutination methodsor immunoblotting methods.

Antibodies according to the invention also have a therapeutic use in thetreatment of fungal infections, for example those just described and maybe used alone or conjugated to an effector molecule, in the latter caseto target the effector molecule, e.g. an anti-fungal agent such asamphotericin B or flacytosine, to the infecting organism. Fortherapeutic use the antibody may be formulated in accordance withconventional procedures, for example with a pharmaceutically acceptablecarrier or excipient, e.g. isotonic saline for administration at anappropriate dosage, depending on the nature of the infection to betreated and the age and condition of the patient.

If desired, mixtures of antibodies may be used for diagnosis ortreatment, for example mixtures of two or more antibodies recognisingdifferent epitopes of a fungal stress protein according to theinvention, and/or mixtures of antibodies of a different class, e.g.mixtures of IgG and IgM antibodies recognising the same or differentepitope(s) of a fungal stress protein of the invention.

The stress proteins according to the invention may be prepared by avariety of processes, for example by protein fractionation fromappropriate fungal cell extracts, using conventional separationtechniques such as ion exchange and gel chromatography andelectrophoresis, or by the use of recombinant DNA techniques, as moreparticularly described in the Examples hereinafter. The use ofrecombinant DNA techniques is particularly suitable for preparingsubstantially pure stress proteins according to the invention.

Thus according to a further aspect of the invention we provide a processfor the production of a fungal stress protein having an amino acidsequence which includes at least the sequence of formula (1) or ananalogue thereof, comprising the steps of (1) culturing a host organismtransformed with a vector including a gene coding for a precursor ofsaid protein, (2) cleaving said precursor to produce said protein and(3) recovering said protein.

Preferably the precursor cleaved in this aspect of the invention is afusion protein comprising at least a portion of a protein produced in atransformed host organism and at least the amino acid sequence offormula (1). Such fusion proteins form a further aspect of theinvention. Desirably the fusion protein includes a protein produced at ahigh level by a transformed host organism. Suitable such proteinsinclude at least a portion of a chloramphenicol acetyltransferase (CAT)protein or, preferably at least a portion of the β-galactosidaseprotein.

According to a still further aspect of the invention we provide a DNAsequence coding for a fungal stress protein having substantially thenucleotide sequence of formula (2) (SEQ.ID.NO:2): ##STR2## andhomologues thereof.

DNA with this sequence may be obtained from fungal genomic DNA asdescribed in the Examples hereinafter.

The DNA sequence according to this aspect of the invention may beincorporated in an expression vector using conventional techniques. Thusin a further aspect of the invention we provide an expression vectorincluding substantially a DNA sequence of formula (2) or a homologuethereof.

The vector may be adapted for use in a given host cell by the provisionof suitable selectable markers, promoters and other control regions asappropriate. Host cells transformed with such vectors form a furtheraspect of the invention. Suitable host organisms include bacteria (e.g.E. coli), and mammalian cells is tissue culture.

The DNA sequence of formula (2) may also be used to design DNA probesfor use in identifying the presence of fungal stress proteins in theinfected state and the invention extends to such DNA probes. Such probesmay also be of use for detecting circulating fungal nucleic acids, forexample using a polymerase chain reaction, as a method of diagnosingfungal infections. The probe may be synthesised using conventionaltechniques and may be immobilised on a solid phase, capable ofimmobilisation on a solid phase, or may be labelled with a detectablelabel.

It will also be appreciated that by suitable epitope mapping, usingconventional procedures, for example as described in Example 2hereinafter peptide fragments of the stress proteins may be identifiedwhich can be chemically synthesised. Synthetic peptide antigens of thistype may be used to raise antibodies for use in diagnosis and/ortherapy, as previously described, or to produce antiseras, e.g.non-specific polyclonal antisera, for use as a vaccine, and as discussedabove form a further aspect of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following description various embodiments of the presentinvention are described with reference to the accompanying drawings towhich:

FIG. 1 shows immunoblots of C. albicans probed with antigen-selectedantibodies.

FIG. 2 shows immunoblots of recombinant lysogen prepared from clone CA-1

FIG. 2B shows immunoblots of recombinant lysogen CA-1 probed withmonoclonal antibody against β-galactosidase

FIG. 2C shows immunoblots of recombinant lysogen CA-1 probed with serafrom patients having antibody to the 47 Kd antigen

FIG. 3 is a comparison of the predicted amino acid sequence of the C.albicans open reading frame (CA-orf) (SEQ.ID.NO.1) with S. cerevisae hsp90 (SEQ.ID.NO:3)

FIG. 4 shows immunoblots of C. albicans in the yeast phase probed withrabbit antiserum raised against LKVIRKNIVKKMIE-Cys-KLH (SEQ ID NO:8)

FIG. 5 shows immunoblots of C. albicans in the mycelial phase probedwith rabbit antiserum raised against LKVIRKNIVKKMIE-Cys-KLH (SEQ IDNO:8)

DESCRIPTION OF SPECIFIC EMBODIMENTS

The following Examples illustrate the invention, in which Example 1describes the preparation of a stress protein according to theinvention; Example 2 describes the epitope mapping of a stress proteinaccording to the invention, the preparation of particular peptideepitopes and their use in detecting Candida infections; Example 3describes the preparation of a monoclonal antibody against a particularepitope; and Example 4 describes the use of a particular monoclonalantibody raised against an epitope of a stress protein according to theinvention! to protect against Candida infection in mice.

EXAMPLE 1

Strain and culture conditions

A fully characterised strain of C. albicans (serotype A, morphotype A1,biotype 157), responsible for the first London hospital outbreak ofsystemic candidiasis Burnie, J. P. et al, Brit. Med. J., 290, 746-748(1985)! was grown at 37° C. overnight with seration in 2% glucose broth.

Preparation and screening of genomic library

Genomic DNA was prepared by the method of Wills et al J. Bacteriol. 157,918-924 (1984)!, as modified by Scherer and Stevens Proc. Natn. Acad.Sci. USA 85, 1452-1456, (1988)!. It was partially digested with EcoRIand fragments 2-7 kilobase pairs long were cloned into the unique EcoRIrestriction enzyme site of the expression vector lambda gt 11 Huynh, T.et al in DNA Cloning Techniques: A Practical Approach, Glover, D. Ed,pp. 49-78, IRL Press, Oxford (1985), and Young, R. A. and Davis, P. W.,Proc. Natn. Acad. Sci. USA 80, 1194-1198, (1983)!. The library wasscreened with rabbit antiserum raised against soluble candidal antigensproduced by fragmenting C. albicans yeast cells at -20° C. in an X-pressMatthews, R. C. et al, J. Clin. Microbiol. 25, 230-237 (1987)-(1)!.Immunoblotting against C. albicans, as previously described Matthews, R.C. et al, Lancet ii, 1415-1418, (1984)-2); Matthews, R. C. et al J.Clin. Microbiol. ibid!, confirmed that the antiserum contained hightitre antibody to many antigens including the 47 KD antigen. Fivepositive clones were identified by screening approximately 10⁵ plaques.

Characterisation of positive clones

The epitope expressed by each of the positive clones was identified byantigen-selection as described by Lyon et al Proc. Natn. Acad. Sci. USA,83, 2989-2993, (1986)!. For this the polyspecific rabbit antiserum wasaffinity purified against positive recombinant plaques and the boundantibody, eluted with glycine buffer pH 2.8, screened against animmunoblot of C. albicans Matthews, R. C. et al. (1), (2) ibid!.Lysogens were prepared in E. coli Y1089 as described by Huynh et alibid!. To determine whether expression of the fusion protein was underthe control of the lac Z promoter, lysates of the recombinant lysogenswere examined, by immunoblotting, after: 1) heat-induction at 45° C.followed by growth at 37° C. for 60 min. with 10 mM isopropylβ-d-thiogalactoside (EPIG); 2) heat induction following by growth at 30°C. without IPIG; and 3) growth at 32° C. in the presence of 10 mM IPTG.

Immunoblots of recombinant lysogens were examined for reactivity of thefusion protein with: 1) rabbit candidal antiserum (diluted 1:2 in 3%bovine serum albumin in buffered saline); 2) a monoclonal antibody toβ-galactosidase, obtained commercially from Promega Biotec, Liverpool(1:5,000 dilution); and 3) sera (1:10) from patients with antibody tothe 47 KD antigen, including five patient with AIDS and one patientrecovering from systemic candidiasis who seroconverted to the 47 KDantigen; sera from five HIV antibody-positive patients with no evidenceof candidiasis were use as controls.

DNA sequencing and analysis

Restriction enzyme mapping inserts from the five positive clonesidentified an overlapping region, suggesting that the epitopes expressedby each clone were encoded by a single genomic segment. This region fromclone CA-1, extending 2 KB from the 5' termini of the insert, wassubcloned into the EcoRI site of pUC19. It was sequenced by the dideoxychain termination method of Sanger et al Proc. Natn. Acad. Sci. USA 745463-5467 (1977)!. Reading frame analysis revealed a single open readingframe (CA-orf) extending from the EcoRI cloning site. The FASTAprogramme (Pearson, W. E. and Lipman, D. J., Proc. Natn. Acad. Sci. USA,85, 2444-2448, (1988)!was used to compare the predicted polypeptide withthe PIR protein database.

RESULTS

All five clones expressed epitopes which cross-reacted with the 47 KDantigen and a 92 KD antigen of C. albicans (FIG. 1). Immunoblots ofrecombinant lysogens, grown with and without IPTG, demonstrated thatexpression was under the regulation of the lac promotor (FIG. 2A). Thecloned antigen was fused to β-galactosidase. The fusion protein producedby clone CA-1 had an elevated molecular weight, Mr 160 KD, compared tonative β-galactosidase (Mr 116 KD) (FIG. 2B). A lower Mr band was alsoseen reacting with the anti-galactosidase monoclonal antibody,indicating the fusion protein was inherently less stable than nativeβ-galactosidase.

As well as the rabbit candidal antiserum, the fusion protein alsoreacted with sera from AIDS patients with antibody to the 47 KD antigen,but not HIV antibody positive patients without this antibody. A patientwith systemic candidiasis who seroconverted to the 47 KD antigen alsoseroconverted to the fusion protein (FIG. 2C). The patients' sera, whichhad not been absorbed with E. coli, reacted with several other bands inthe recombinant lysogens and the λgt 11 control, and therefore thesebands were considered to be E. coli antigens.

Nucleotide analysis of the insert DNA from clone CA-1 revealed a singlepartial open reading frame (orf) which continued in from the cloningsite and coded for a polypeptide of 395 amino acids (Mr 45 KD). This orfwas in phase with the β-galactosidase gene. Since fusion withβ-galactosidase (Mr 116 KD) results in removal of the C-terminal 19residues from the lac Z gene, the calculated size of the fusion proteinwas 159 KD. This agrees with the estimated value of 160 KD for thefusion protein produced by clone CA-1.

A database search, with the polypeptide sequence derived from this orf,revealed significant (>45%) homologies with heat shock proteins (haps)from Drosophila Blackman, R. K. and Meselson, M. (1986) J. Mol. Biol.188, 499-515! and chickens Kxlomaa, M. S., et al (1986) Biochemistry 25,6244-6251! and microsomal glucose-regulated proteins (grps) fromhamsters Sorger, P. E. and Pelham, H. R. B., (1987) J. Mol. Biol. 194,341-344! and mice Smith, M. J. and Koch, G. L. E., (1987) J. Mol. Biol.194, 345-347!. The most extensive sequence similarity was found with theyeast hap 90 protein of Saccharomyces cerevisiae Farrelly, F. W. andFinkelstein, D. B. (1984) J. Biol. Chem. 259, 5745-5751!, with 83.5%identity in the 395 amino acid overlap (FIG. 3).

The following figures are referred to above. In the figures:

FIG. 1 shows immunoblots of C. albicans probed with: antigen-selectedantibodies from two of the positive clones showing cross-reactivity withthe 92 KD and 47 KD antigens, and weakly, two intermediate components(tracks a and b); negative control eluate from non-recombinant plaques(c); rabbit candidal antiserum (d); AIDS patient's serum containing hightitre antibody to the 47 KD antigen (e).

FIG. 2A shows immunoblots of recombinant lysogen prepared from cloneCA-1, probed with rabbit candidal antiserum, showing the 160 KD fusionprotein is present when heat induction at 45° C. is followed by growthat 37° C. for 60 min. with 10 mM IPTG (tracks c and d, showing twodifferent lysogenic preparations). It is not produced when lysogens aregrown at 32° C. (track b) or heat-induced but grown without IPTG (trackse and f). Molecular weight markers (KD) shown in track a.

FIG. 2B shows immunoblots of recombinant lysogen CA-1 probed withmonoclonal antibody against β-galactosidase. The 160 KD fusion proteinand breakdown product are shown after growth with IPTG (track a), andwithout IPTG (track b); a non-recombinant λgt11 lysogen, grown with andwithout IPTG shows the position of native β-galactosidase (Mr 116 KD)(tracks c and d).

FIG. 2C shows immunoblots of recombinant lysogen CA-1 probed with serafrom patients having antibody to the 47 KD antigen. Serum from an AIDSpatient with antibody to the 47 KD antigen cross-reacting with 160 KDfusion protein (track a); and early (b) and late (c) sera from a patientrecovering from systemic candidiasis who seroconverted to the 47 KDantigen, showing seroconvertion to the 160 KD fusion protein.

FIG. 3 is a comparison of the predicted amino acid sequence of the C.albicans open reading frame (CA-orf) (SEQ ID NO:1) with S. cerevisaehsp90 (SCHS90) (SEQ ID NO:3). Over the CA-orf sequence they showed >83%direct homology (:) or >98% conserved homology (.).

EXAMPLE 2

The sequenced carboxy end of a C. albicans stress protein according tothe invention was epitope mapped using the method described by Geysen H.M. et al in Journal of Immunological Methods 102, 259-274 (1987). Thistechnique involves the synthesis of large numbers of overlappingpeptides and identifies continuous antigenic peptides on a proteinantigen. It will not detect carbohydrate or discontinuous epitopes. Byway of example further details of two of these epitopes are given below:

1. STDEPAGESA (SEQ ID NO:4)

This epitope occurs just before the carboxy terminal 5 amino acidresidues of the sequence of formula (1). It reacted with: (1)hyperimmune sera from 2 out of 3 rabbits immunised with C. albicanspressate as described in Burnie J. P., et al J. Clin. Pathol. 38,701-106 (1985)!; (2) sera from 3 out of 5 patients with systemiccandidiasis, two of whom seroconverted to this epitope; (3) pooled serafrom 4 patients who were HIV antibody positive who have antibody to the47 Kd antigen--see Matthews R. C., et al Lancet ii; 263-266 (1988)!. Itdid not react with sera from 7 normal control patients or a patient withchronic mucocutaneous candidiasis (CMC).

This epitope was synthesised and conjugated to keyhole limpethaemocyanin (KLH) via the terminal cysteine to give KLH-Cys-STDEPAGESA(SEQ ID NO:9). A rabbit polyclonal antiserum raised against this, onimmunoblotting against C. albicans, recognised a heat-inducible antigenat about 92 Kd (present AT 37° C. but not 23° C.) and a constitutiveantigen at about 40 Kd (present when grown at 23° C. or 37° C.). It didnot detect the 47 Kd antigen. It did not cross-react with S. cerevisiaeor A. fumigatus.

2. LSREM-LKVIRK

These two epitopes are situated close to each other, 316-332 amino acidresidues from the carboxy end of the sequence of formula (1). The LSREM(SEQ ID NO:5) epitope reacted with; (1) 2 out of 3 rabbit hyperimmunecandidal antisera; (2) 4 out of 5 sera from patients with systemiccandidiasis, one of whom seroconverted to it; (3) pooled sera from 4 HIVantibody positive patients and (4) serum from the CMC patient. It didnot react with sera from 7 negative control patients. A rabbit immunisedwith KLH-Cys-LPLNLSREML (SEQ ID NO:10) failed to produce antibody to it.

The LKVIRK (SEQ ID NO:6) epitope was specifically detected by infectedpatients' sera. None of the hyperimmune rabbit sera recognized it norany of the 7 negative control patients. All five patients with systemiccandidiasis had antibody to this epitope and in two case, where serialsera were available, seroconverted to it. The pooled sera from 4 HIVantibody positive patients and the patient with CMC also recognised thisepitope. A rabbit polyclonal antiserum raised against the epitope(LKVIRKNIVKKMIE-Cys-KLH, (SEQ ID NO:8) recognised both the 47 Kd antigenand the antigen of about 92 Kd on immunoblots of various strains of C.albicans (including serotypes A and B) in both the yeast and mycelialphase (FIGS. 4 and 5). It also recognised the fusion protein produced bya positive clone. Cross-absorption with the synthesised peptide epitoperemoved this antibody activity. The antibody also recognised antigens onimmunoblots of (1) Candida parapsilosis--giving a band at about 52 Kd(an antigen of this size was previously reported by Belder M. A., et alEuropean heart Journal 10, 858-862 1989!); (2) A. fumigatus--givingbands at about 88 Kd, 51 Kd and 40 Kd these antigens have previouslybeen reported, Matthews R. C., et al J. Clin. Pathol. 38, 1300-1303(1985)! and (3) S. cerevisiae--at about 84 Kd and 45 Kd, compatible withS. cerevisiae hsp 90. This therefore suggests the presence of a stressprotein according to the invention in A. fumigatus of about 88 Kd.

In FIGS. 4 and 5 referred to above, the following is shown:

FIG. 4 shows immunoblots of C. albicans in the yeast phase probed withrabbit antiserum raised against LKVIRKNIVKKMIE-Cys-KLH (SEQ ID NO:8)both before (PRE) and after (POST) cross-absorption with this peptide.Molecular weight markers shown on left hand side. Lane number 1,outbreak strain of C. albicans Burnie J. P., et al B.M.J. 290, 746-748(1985)!; lane no. 2, C. albicans strain MCTC3153 (serotype A); lane no.3, C. albicans strain NCTC3156 (serotype B). The 47 Kd antigen and theantigen at about 92 Kd are arrowed. Notice these antigens are missingafter cross-absorption.

FIG. 5 Lanes 1-3 are as in FIG. 5 but with the yeast in the mycelialphase. Probed with the same antiserum as in FIG. 4, pre and postcross-absorption with the peptide. Lane no. 4 is a positive cloneshowing the fusion protein disappearing after cross-absorption, as dothe 47 Kd and 92 Kd antigens in lanes no. 1-3.

Antibody raised against the peptide STDEPAGESA (SEQ ID NO:4) orLKVIRKNIVKKMIE (SEQ ID NO:7) was used to examine the sera of patientswith systemic candidiasis as follows:

Antigen was detected by dot-blotting serum directly onto nitrocellulosemembrane. Briefly, 100 μl of antiserum was loaded into each well of aBio-Dot microfiltration apparatus (Biorad laboratories). This had beenpreviously loaded with a sheet of nitrocellulose membrane prewashed inpH 7.5 Tris buffered saline (TBS). The sample was allowed to drainthrough under gravity. Each well was loaded with a further 100 μl of TBSwhich drains under gravity control. This is followed by 2×100 μl TBSdrained by vacuum. The membrane was then blocked in 3% BSA in TBS at 4°C. overnight. In the morning it was incubated with 30 μl of theappropriate rabbit serum in 3% BSA in TBS for 2 hours at roomtemperature. It was then washed in 20 mM Tris, 500 mM NaCl, 0.05% Tween20, pH 7.5 for 30 minutes and incubated with alkaline phosphataseconjugated antirabbit (1:1000) (Sigma) for 1 hour at room temperature.It was washed again and stained for 15 minutes at room temperature in afreshly prepared and filtered mixture of equal volumes of naphthol AS-MXphosphate (Sigma; 0.4 mg/ml in distilled water) and fast red TR salt(Sigma; 6 mg/ml in 0.2M Tris, pH 8.2). Antibody intensity was comparedby eye with that of controls whose density had been measured previouslywith a Joyce Loebl scanning densitometer. The results were classified aspositive or trace according to the criteria of Matthews, R. C. andBurnie, J. P. (J. Clin. Micro., 26: 459-463 (1988).

Three groups of sera were examined. The Control sera came from patientswho had been screened for systemic candidiasis but where there was noclinical or cultural evidence for the infection. The second group waspatients who were colonized at clinically significant sites (intravenouslines, wound, faeces, urine and vagina) and where there was no evidenceof dissemination. The third group consisted of either suspected orproven cases. In the suspected cases a clinically significant pyrexiaresolved on systemic amphotericin B therapy. The patients wereneutropenic and although some cultures were positive for Candidaalbicans this was insufficient to prove the infection. In the provencases there was either cultural and histological evidence from a deepsite at autopsy or two sets of positive blood cultures taken fromseparate sites on two different occasions during life.

The sera were examined against antibody raised against the peptideSTDEPAGESA (SEQ ID NO:4) or the peptide LKVIRKNIVKKMIE (SEQ ID NO:7).The former detected (trace or positive response) 90.4% of cases. Thelatter detected 88.2% of proven and all the suspected cases. There wasalso a positive response in four patients where a localized infectionrequired treatment with systemic amphotericin B. Both antibodiesdetected circulating candidal antigen specific to disseminatedcandidiasis.

    ______________________________________                                        Results of Dot-Blotting                                                                            Nil      Trace  Positive                                 ______________________________________                                        1.  Antibody against STDEPAGESA                                                   (SEQ ID NO. 4)                                                                Controls             65                                                       Colonized            6        1                                               Systemically infected                                                                              2        8    11                                         (proven)                                                                  2.  Antibody against LKVIRKNIVKKMIE                                               (SEQ ID NO. 7)                                                                Controls             404                                                      Colonized            10       5    .sup. 4.sup.a                              Systemically infected                                                         (proven)             6        9    36                                         (suspected)                   4     6                                     ______________________________________                                         .sup.a required systemic therapy for N line infection (1), urinary tract      infection (2) and wound infection (1) with amphotericin B.               

EXAMPLE 3

A murine monoclonal antibody was raised against LKVIRKNIVKKMIE-Cys-KLH(SEQ ID NO:8) (see Example 2). Balb/c and CBA×Balb/c F1 mice wereinjected subcutaneously with 50 μg of immunogen in sterile completeFreund's Adjuvant and thereafter at intervals of 14 days,intraperitoneally with 50 μg immunogen in Incomplete Freund's Adjuvantuntil seroconvertion.

Fusion was performed 4 days after a final immunization of 50 μgimmunogen intravenously in sterile physiological saline. Fusion,hybridoma screening, clonal selection and antibody analysis wereperformed according to standard protocols, essentially as described byde St. Groth S. F. and Scheidegger D., J. Immunol. Methods 35, 1-21(1980). Selected hybridomas were screened for activity against the C.albicans 47 Kd antigen by immunoblotting against C. albicans. Positivehybridomas were re-cloned and re-assayed.

A novel hybridoma cell line (CA-STR7-1) was produced which recognisedboth the C. albicans 47 Kd antigen and the antigen of approximately 92Kd on immunoblots of C. albicans grown at 37° C., in both the yeast andmycelial forms. At 23° C. the 47 Kd antigen was visible but not the 92Kd antigen with this monoclonal antibody.

EXAMPLE 4

A monoclonal antibody was raised against the peptide STDEPAGESA (SEQ IDNO:4) using the method of Example 3, and was used in a mouse model todetermine whether it had a protective effect against challenge with alethal dose of C. albicans. The mouse model was as follows:

Male Balb C mice of average weight 2.5 g were injected i.v. withdilutions of a standardized batch of frozen C. albicans. The injectedvolume was 100 μl via the lateral tail vein. The following mortality wasobserved at the doses and times shown:

    ______________________________________                                                 Mortality at                                                         Dose       18 hrs        24 hrs  n                                            ______________________________________                                        5 + 10.sup.8 cells                                                                       89%           100%    54                                           1 + 10.sup.8 cells                                                                       76%           100%    48                                           5 + 10.sup.7 cells                                                                        0%            0%     24                                           ______________________________________                                    

The viability of injected cells from frozen aliquats was 30% andtherefore the dose could be adjusted downwards.

A challenge of 1×10⁸ C. albicans cells was used as described above inmice which had been pre-treated with the antibody against STDEPAGESA(SEQ ID NO:4) by prior injection as follows:

    ______________________________________                                                   Mortality at    "Protective" Agent                                         n    24 hrs  48 hrs    injected volume                                ______________________________________                                        STDEPFAESA                                                                              6      66%       83.3% 0.2 ml 1 hr prior to                         (SEQ ID NO. 4)                   challenge                                    Mab L2    6      100%    --      0.2 ml 1 hr prior to                                                          challenge                                    Patient 1 6      50%     50%     0.5 ml 30 min prior to                                                        challenge                                    Patient 2 7        42.8% 42.8%   0.5 ml 30 min prior to                                                        challenge                                              5      40%     40%     0.5 ml 30 min prior to                                                        challenge                                    Normal Human                                                                            6      100%    --      0.5 ml 30 min prior to                       Serum                            challenge                                    IG fraction                                                                             6      50%     66%     0.5 ml 30 min prior to                       Patient 2                        challenge                                    IG fraction                                                                             6      100%    --      0.5 ml 30 min prior to                       normal human                     challenge                                    serum                                                                         ______________________________________                                    

"Patient 1" and "Patient 2" was serum from patients who developedsystemic Canidada infections, developed an antibody response andrecovered.

"IG fraction" was an immunoglobulin fraction obtained by 50% ammoniumsulphate precipitation, dialysed overnight v PBS and resuspended in thesame volume as the original serum.

Mab L2 was an irrelevant IgG antibody.

Conclusion

The monoclonal antibody raised against STDEPAGESA (SEQ ID NO:4) produced33% survival at 24 hrs in animals challenged with a lethal dose of C.albicans, whereas the irrelevant IgG and normal human serum produced noprotection. The serum from the previously infected patients produced 55%protection at 24 hrs.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 10                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 395 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GluPheArgAlaIleLeuPheValProLysArgAlaProPheAspAla                              151015                                                                        PheGluSerLysLysLysLysAsnAsnIleLysLeuTyrValArgArg                              202530                                                                        ValPheIleThrAspAspAlaGluGluLeuIleProGluTrpLeuSer                              354045                                                                        PheIleLysGlyValValAspSerGluAspLeuProLeuAsnLeuSer                              505560                                                                        ArgGluMetLeuGlnGlnAsnLysIleLeuLysValIleArgLysAsn                              65707580                                                                      IleValLysLysMetIleGluThrPheAsnGluIleSerGluAspGln                              859095                                                                        GluGlnPheAsnGlnPheTyrThrAlaPheSerLysAsnIleLysLeu                              100105110                                                                     GlyIleHisGluAspAlaGlnAsnArgGlnSerLeuAlaLysLeuLeu                              115120125                                                                     ArgPheTyrSerThrLysSerSerGluGluMetThrSerLeuSerAsp                              130135140                                                                     TyrValThrArgMetProGluHisGlnLysAsnIleTyrTyrIleThr                              145150155160                                                                  GlyGluSerIleLysAlaValGluLysSerProPheLeuAspAlaLeu                              165170175                                                                     LysAlaLysAsnPheGluValLeuPheMetValAspProIleAspGlu                              180185190                                                                     TyrAlaMetThrGlnLeuLysGluPheGluAspLysLysLeuValAsp                              195200205                                                                     IleThrLysAspPheGluLeuGluGluSerAspGluGluLysAlaAla                              210215220                                                                     ArgGluLysGluIleLysGluTyrGluProLeuThrLysAlaLeuLys                              225230235240                                                                  AspIleLeuGlyAspGlnValGluLysValValValSerTyrLysLeu                              245250255                                                                     ValAspAlaProAlaAlaIleArgThrGlyGlnPheGlyTrpSerAla                              260265270                                                                     AsnMetGluArgIleMetLysAlaGlnAlaLeuArgAspThrThrMet                              275280285                                                                     SerSerTyrMetSerSerLysLysThrPheGluIleSerProSerSer                              290295300                                                                     ProIleIleLysGluLeuLysLysLysValGluThrAspGlyAlaGlu                              305310315320                                                                  AspLysThrValLysAspLeuThrThrLeuLeuPheAspThrAlaLeu                              325330335                                                                     LeuThrSerGlyPheThrLeuAspGluProSerAsnPheAlaHisArg                              340345350                                                                     IleAsnArgLeuIleAlaLeuGlyLeuAsnIleAspAspAspSerGlu                              355360365                                                                     GluThrAlaValGluProGluAlaThrThrThrAlaSerThrAspGlu                              370375380                                                                     ProAlaGlyGluSerAlaMetGluGluValAsp                                             385390395                                                                     (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1200 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GAATTCAGAGCTATCTTGTTTGTTCCAAAGAGAGCTCCATTTGATGCCTTTGAATCCAAG60                AAGAAGAAGAACAACATCAAATTATACGTCCGTAGAGTGTTTATCACTGATCATGCTGAA120               GAGTTGATTCCAGAATGGTTAAGTTTCATCAAGGGGGTTGTCGATTCCGAAGACTTGCCA180               TTGAACTTGTCCAGAGAAATGTTGCAACAAAACAAGATTTTGAAAGTTATCAGAAAGAAC240               ATTGTCAAAAAGATGATTGAAACTTTCAATGAAATCTCTGAAGACCAAGAGCAATTCAAC300               CAATTCTACACTGCTTTCTCCAAGAACAYCAAAYYHHHYAYYCAYHAAHAYHCYCAAAAC360               AGACAATCTTTGGCTAAATTGTTGAGATTCTACTCTACCAAATCTTCTGAAGAAATGACT420               TCCTTGTCTGACTACGTTACTAGAATGCCAGAACACCAAAAGAATATCTACTACATCACT480               GGTGAATCCATCAAAGCCGTTGAAAAATCACCATTCTTGGATGCCTTGAAAGCTAAGAAC540               TTTGAAGTCTTGTTCATGGTGGATCCAATCGATGAATATGCCATGACTCAATTGAAGGAA600               TTTGAAGACAAGAAATTGGTTGATATTACCAAAGACTTTGAATTGGAAGAAAGTGACGAA660               GAAAAAGCTGCTAGAGAAAAGGAAATCAAAGAATACGAACCATTGACCAAAGCTTTGAAA720               GATATTCTTGGTGSTCAAGTTGAAAAAGTTGTTGTTTCCTACAAACTTGTTGATGCTCCA780               GCTGCCATTSGAACTGGTCAATTTGGTTGGTCTGCCAATATGGAAAGAATCATGAAGGCT840               CAAGCTTTGAGAGACACCACCATGTCTTCTTACATGTCCTCTAAGAAGACCTTTGAAATT900               TCTCCATCTTCCCCAATTATCAAGGAATTCAAGAAGAAAGTTGAAACCGATGGAGCTGAA960               GACAAGACCGTTAAGGACTTGACCACTTTGTTGTTTGATACTGCATTGTTGACTTCTGGT1020              TTCACCTTGGACGAACCATCCAACTTTGCCCACAGAATTAACAGATTGATTGCCTTGGGA1080              TTGAATATTGACGATGATTCAGAAGAAACTGCTATTGAACCTGAAGCTACTACTACTGCC1140              TCAACTGACGAACCAGCTGGAGAATCTGCTATGGAAGAAGTTGATTAAACACCAGAAGGG1200              (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 394 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GluPheArgAlaIleLeuPheIleProLysArgAlaProPheAspLeu                              151015                                                                        PheGluSerLysLysLysLysAsnAsnIleLysLeuTyrValArgArg                              202530                                                                        ValPheIleThrAspGluAlaGluAspLeuIleProGluTrpLeuSer                              354045                                                                        PheValLysGlyValValAspSerGluAspLeuProLeuAsnLeuSer                              505560                                                                        ArgGluMetLeuGlnGlnAsnLysIleLeuLysValIleArgLysAsn                              65707580                                                                      IleValLysLysLeuIleGluAlaPheAsnGluIleAlaGluAspSer                              859095                                                                        GluGlnPheGluLysPheTyrSerAlaPheSerLysAsnIleLysLeu                              100105110                                                                     GlyValHisGluAspThrGlnAsnArgAlaAlaLeuAlaLysLeuLeu                              115120125                                                                     ArgTyrAsnSerThrLysSerValAspGluLeuThrSerLeuSerAsp                              130135140                                                                     TyrValThrArgMetProGluHisGlnLysAsnIleTyrTyrIleThr                              145150155160                                                                  GlyGluSerLeuLysAlaValGluLysSerProPheLeuAspAlaLeu                              165170175                                                                     LysAlaLysAsnPheGluValLeuPheLeuThrAspProIleAspGlu                              180185190                                                                     TyrAlaPheThrGlnLeuLysGluPheGluAspLysThrLeuValAsp                              195200205                                                                     IleThrLysAspPheGluLeuGluGluThrAspGluGluLysAlaGlu                              210215220                                                                     ArgGluLysGluIleLysGluTyrGluProLeuThrLysAlaLeuLys                              225230235240                                                                  GluIleLeuGlyAspGlnValGluLysValValValSerTyrLysLeu                              245250255                                                                     LeuAspAlaProAlaAlaIleArgThrGlyGlnPheGlyTrpSerAla                              260265270                                                                     AsnMetGluArgIleMetLysAlaGlnAlaLeuArgAspSerSerMet                              275280285                                                                     SerSerTyrMetSerSerLysLysThrPheGluIleSerProLysSer                              290295300                                                                     ProIleIleLysGluLeuLysLysArgValAspGluGlyGlyAlaGln                              305310315320                                                                  AspLysThrValLysAspLeuThrLysLeuLeuTyrGluThrAlaLeu                              325330335                                                                     LeuThrSerGlyPheSerLeuAspGluProThrSerPheAlaSerArg                              340345350                                                                     IleAsnArgLeuIleSerLeuGlyLeuAsnIleAspGluAspGluGlu                              355360365                                                                     ThrGluThrAlaProGluAlaSerThrAlaAlaProValGluGluVal                              370375380                                                                     ProAlaAspThrGluMetGluGluValAsp                                                385390                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       SerThrAspGluProAlaGlyGluSerAla                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       LeuSerArgGluMet                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       LeuLysValIleArgLys                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       LeuLysValIleArgLysAsnIleValLysLysMetIleGlu                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       LeuLysValArgLysAsnIleValLysLysMetIleGluCys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CysSerThrAspGluProAlaGlyGluSerAla                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CysLeuProLeuAsnLeuSerArgGluMetLeu                                             1510                                                                          __________________________________________________________________________

We claim:
 1. A method for diagnosing the Candida infection wherein said method comprises:a) combining a nucleic acid sample with a probe having the nucleotide residue sequence represented by SEQ ID NO:2 wherein said sample is suspected of containing Candida nucleic acid; b) allowing for the formation of a hybridization product between the probe and said target nucleic acid under conditions which only permit the formation of a hybridization product between the probe and said target nucleic acid; and c) detecting the presence of said hybridization product.
 2. The method of claim 1 further comprising amplifying the target nucleic acid prior to formation of said hybridization product.
 3. The method of claim 2 wherein said amplification is achieved by performing a polymerase chain reaction.
 4. The method of claim 1 wherein the probe is detectably labeled. 